Novel human kinases and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/239,821, which was filed on Oct. 12, 2000, and isherein incorporated by reference in its entirety.

INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding proteinssharing sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or over express the disclosed genes,antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed genes that can be used for diagnosis, drug screening, clinicaltrial monitoring, the treatment of diseases and disorders, and cosmeticor nutriceutical applications.

BACKGROUND OF THE INVENTION

[0003] Kinases mediate the phosphorylation of a wide variety of proteinsand compounds in the cell. Along with phosphatases, kinases are involvedin a range of regulatory pathways. Given the physiological importance ofkinases, they have been subject to intense scrutiny and are proven drugtargets.

SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode novel human proteins andthe corresponding amino acid sequences of these proteins. The novelhuman proteins (NHPS) described for the first time herein sharestructural similarity with animal kinases, including, but not limitedto, serine-threonine kinases, calcium/calmodulin-dependent proteinkinases, and mitogen activated kinases. Accordingly, the described NHPsencode novel kinases having homologues and orthologs across a range ofphyla and species.

[0005] The novel human polynucleotides described herein, encode openreading frames (ORFs) encoding proteins of 766 and 765 amino acids inlength (see respectively SEQ ID NOS: 2 and 4).

[0006] The invention also encompasses agonists and antagonists of thedescribed NHPS, including small molecules, large molecules, mutant NHPs,or portions thereof, that compete with native NHP, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stemcells (“ES cells”) lines that contain gene trap mutations in a murinehomolog of at least one of the described NHPs. When the unique NHPsequences described in SEQ ID NOS:1-4 are “knocked-out” they provide amethod of identifying phenotypic expression of the particular gene aswell as a method of assigning function to previously unknown genes. Inaddition, animals in which the unique NHP sequences described in SEQ IDNOS:1-4 are “knocked-out” provide a unique source in which to elicitantibodies to homologous and orthologous proteins that would have beenpreviously viewed by the immune system as “self” and therefore wouldhave failed to elicit significant antibody responses. To these ends,gene trapped knockout ES cells have been generated in murine homologs ofthe described NHPs.

[0007] Additionally, the unique NHP sequences described in SEQ IDNOS:1-4 are useful for the identification of protein coding sequence andmapping a unique gene to a particular chromosome. These sequencesidentify biologically verified exon splice junctions as opposed tosplice junctions that may have been bioinformatically predicted fromgenomic sequence alone. The sequences of the present invention are alsouseful as additional DNA markers for restriction fragment lengthpolymorphism (RFLP) analysis, and in forensic biology.

[0008] Further, the present invention also relates to processes foridentifying compounds that modulate, i.e., act as agonists orantagonists, of NHP expression and/or NHP activity that utilize purifiedpreparations of the described NHPs and/or NHP product, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0009] The Sequence Listing provides the sequence of the novel humanORFs encoding the described novel human kinase proteins.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The NHPs described for the first time herein are novel proteinsthat are expressed in, inter alia, human cell lines and human fetalbrain, brain, pituitary, spinal cord, testis, adipose, and esophaguscells. The described sequences were compiled from sequences available inGENBANK, and cDNAs generated from skeletal muscle, adipose, pituitary,cerebellum, and brain MRNA (Edge Biosystems, Gaithersburg, Md.).

[0011] The present invention encompasses the nucleotides presented inthe Sequence Listing, host cells expressing such nucleotides, theexpression products of such nucleotides, and: (a) nucleotides thatencode mammalian homologs of the described genes, including thespecifically described NHPs, and the NHP products; (b) nucleotides thatencode one or more portions of an NHP that correspond to functionaldomains, and the polypeptide products specified by such nucleotidesequences, including but not limited to the novel regions of any activedomain(s); (c) isolated nucleotides that encode mutant versions,engineered or naturally occurring, of the described NHPs in which all ora part of at least one domain is deleted or altered, and the polypeptideproducts specified by such nucleotide sequences, including but notlimited to soluble proteins and peptides in which all or a portion ofthe signal sequence is deleted; (d) nucleotides that encode chimericfusion proteins containing all or a portion of a coding region of a NHP,or one of its domains (e.g., a receptor/ligand binding domain, accessoryprotein/self-association domain, etc.) fused to another peptide orpolypeptide; or (e) therapeutic or diagnostic derivatives of thedescribed polynucleotides such as oligonucleotides, antisensepolynucleotides, ribozymes, dsRNA, or gene therapy constructs comprisinga sequence first disclosed in the Sequence Listing. As discussed above,the present invention includes: (a) the human DNA sequences presented inthe Sequence Listing (and vectors comprising the same) and additionallycontemplates any nucleotide sequence encoding a contiguous NHP openreading frame (ORF) that hybridizes to a complement of a DNA sequencepresented in the Sequence Listing under highly stringent conditions,e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.lxSSC/0.1%SDS at 68° C. (Ausubel et al., eds., 1989, Current Protocols inMolecular Biology, Vol. I, Green Publishing Associates, Inc., and JohnWiley & sons, Inc., New York, at p. 2.10.3) and encodes a functionallyequivalent expression product. Additionally, contemplated are anynucleotide sequences that hybridize to the complement of the DNAsequence that encode and express an amino acid sequence presented in theSequence Listing under moderately stringent conditions, e.g., washing in0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yet stillencode a functionally equivalent NHP product. Functional equivalents ofa NHP include naturally occurring NHPs present in other species andmutant NHPs whether naturally occurring or engineered (by site directedmutagenesis, gene shuffling, directed evolution as described in, forexample, U.S. Pat. Nos. 5,837,458 or 5,723,323, both of which are hereinincorporated by reference). The invention also includes degeneratenucleic acid variants of the disclosed NHP polynucleotide sequences.

[0012] Additionally contemplated are polynucleotides encoding NHP ORFs,or their functional equivalents, encoded by polynucleotide sequencesthat are about 99, 95, 90, or about 85 percent similar to correspondingregions of SEQ ID NO:1 (as measured by BLAST sequence comparisonanalysis using, for example, the GCG sequence analysis package usingdefault parameters).

[0013] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHP encoding polynucleotides. Such hybridizationconditions can be highly stringent or less highly stringent, asdescribed above. In instances where the nucleic acid molecules aredeoxyoligonucleotides (“DNA oligos”), such molecules are generally about16 to about 100 bases long, or about 20 to about 80, or about 34 toabout 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of sequencefirst disclosed in the Sequence Listing. Such oligonucleotides can beused in conjunction with the polymerase chain reaction (PCR) to screenlibraries, isolate clones, and prepare cloning and sequencing templates,etc.

[0014] Alternatively, such NHP oligonucleotides can be used ashybridization probes for screening libraries, and assessing geneexpression patterns (particularly using a micro array or high-throughput“chip” format). Additionally, a series of the described NHPoligonucleotide sequences, or the complements thereof, can be used torepresent all or a portion of the described NHP sequences. Anoligonucleotide or polynucleotide sequence first disclosed in at least aportion of one or more of the sequences of SEQ ID NOS: 1-4 can be usedas a hybridization probe in conjunction with a solid supportmatrix/substrate (resins, beads, membranes, plastics, polymers, metal ormetallized substrates, crystalline or polycrystalline substrates, etc.).of particular note are spatially addressable arrays (i.e., gene chips,microtiter plates, etc.) of oligonucleotides and polynucleotides, orcorresponding oligopeptides and polypeptides, wherein at least one ofthe biopolymers present on the spatially addressable array comprises anoligonucleotide or polynucleotide sequence first disclosed in at leastone of the sequences of SEQ ID NOS: 1-4, or an amino acid sequenceencoded thereby. Methods for attaching biopolymers to, or synthesizingbiopolymers on, solid support matrices, and conducting binding studiesthereon are disclosed in, inter alia, U.S. Pat. Nos. 5,700,637,5,556,752, 5,744,305, 4,631,211, 5,445,934, 5,252,743, 4,713,326,5,424,186, and 4,689,405 the disclosures of which are hereinincorporated by reference in their entirety.

[0015] Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-4 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is within a range of between about 8 to about2000 nucleotides. Preferably the probes consist of 60 nucleotides andmore preferably 25 nucleotides from the sequences first disclosed in SEQID NOS:1-4.

[0016] For example, a series of the described oligonucleotide sequences,or the complements thereof, can be used in chip format to represent allor a portion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about-two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

[0017] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-4 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel componentsor gene functions that manifest themselves as novel phenotypes.

[0018] Probes consisting of sequences first disclosed in SEQ ID NOS:1-4can also be used in the identification, selection and validation ofnovel molecular targets for drug discovery. The use of these uniquesequences permits the direct confirmation of drug targets andrecognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the drugs intended target.These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

[0019] As an example of utility, the sequences first disclosed in SEQ IDNOS:1-4 can be utilized in microarrays or other assay formats, to screencollections of genetic material from patients who have a particularmedical condition. These investigations can also be carried out usingthe sequences first disclosed in SEQ ID NOS:1-4 in silico and bycomparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

[0020] Thus the sequences first disclosed in SEQ ID NOS:1-4 can be usedto identify mutations associated with a particular disease and also as adiagnostic or prognostic assay.

[0021] Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in the SEQ ID NOS: 1-4. Alternatively, a restriction mapspecifying the relative positions of restriction endonuclease digestionsites, or various palindromic or other specific oligonucleotidesequences can be used to structurally describe a given sequence. Suchrestriction maps, which are typically generated by widely availablecomputer programs (e.g., the University of Wisconsin GCG sequenceanalysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich.,etc.), can optionally be used in conjunction with one or more discretenucleotide sequence(s) present in the sequence that can be described bythe relative position of the sequence relative to one or more additionalsequence(s) or one or more restriction sites present in the disclosedsequence.

[0022] For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHP gene antisense molecules, useful, for example,in NHP gene regulation (for and/or as antisense primers in amplificationreactions of NHP gene nucleic acid sequences). With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences can be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

[0023] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including but not limited to 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine,. 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0024] The antisense oligonucleotide can also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0025] In yet another embodiment, the antisense oligonucleotide willcomprise at least one modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

[0026] In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

[0027] Oligonucleotides of the invention can be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), andmethylphosphonate oligonucleotides-can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

[0028] Low stringency conditions are well known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, supra.

[0029] Alternatively, suitably labeled NHP nucleotide probes can be usedto screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms (including, butnot limited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

[0030] For example, the present sequences can be used in restrictionfragment length polymorphism (RFLP) analysis to identify specificindividuals. In this technique, an individual's genomic DNA is digestedwith one or more restriction enzymes, and probed on a Southern blot toyield unique bands for identification (as generally described in U.S.Pat. No. 5,272,057, incorporated herein by reference). In addition, thesequences of the present invention can be used to provide polynucleotidereagents, e.g., PCR primers,. targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e., another DNA sequence that is unique to a particularindividual). Actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments.

[0031] Further, a NHP gene homolog can be isolated from nucleic acidfrom an organism of interest by performing PCR using two degenerate or“wobble” oligonucleotide primer pools designed on the basis of aminoacid sequences within the NHP products disclosed herein. The templatefor the reaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of MRNA prepared from, for example, human or non-humancell lines or tissue known or suspected to express an allele of a NHPgene.

[0032] The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

[0033] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express a NHP gene). A reverse transcription(RT) reaction can be performed on the RNA using an oligonucleotideprimer specific for the most 5′ end of the amplified fragment for thepriming of first strand synthesis. The resulting RNA/DNA hybrid may thenbe “tailed” using a standard terminal transferase reaction, the hybridmay be digested with RNase H, and second strand synthesis may then beprimed with a complementary primer. Thus, cDNA sequences upstream of theamplified fragment can be isolated. For a review of cloning strategiesthat can be used, see e.g., Sambrook et al., 1989, supra.

[0034] A cDNA encoding a mutant NHP sequence can be isolated, forexample, by using PCR. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known. or suspected to be expressed in an individualputatively carrying a mutant NHP allele, and by extending the new strandwith reverse transcriptase. The second strand of the cDNA is thensynthesized using an oligonucleotide that hybridizes specifically to the5′ end of the normal sequence. Using these two primers, the product isthen amplified via PCR, optionally cloned into a suitable vector, andsubjected to DNA sequence analysis through methods well known to thoseof skill in the art. By comparing the DNA sequence of the mutant NHPallele to that of a corresponding normal NHP allele, the mutation(s)responsible for the loss or alteration of function of the mutant NHPgene product can be ascertained.

[0035] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of or known to carry a mutant NHPallele (e.g., a person manifesting a NHP-associated phenotype such as,for example, immune disorders, obesity, high blood pressure, etc.), or acDNA library. can be constructed using RNA from a tissue known, orsuspected, to express a mutant NHP allele. A normal NHP gene, or anysuitable fragment thereof, can then be labeled and used as a probe toidentify the corresponding mutant NHP allele in such libraries. Clonescontaining mutant NHP sequences can then be purified and subjected tosequence analysis according to methods well known to those skilled inthe art.

[0036] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant NHP allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against a normal NHP product, as described below. (For screeningtechniques, see, for example, Harlow, E. and Lane, eds., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, Cold SpringHarbor.)

[0037] Additionally, screening can be accomplished by screening withlabeled NHP fusion proteins, such as, for example, alkalinephosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In caseswhere a NHP mutation results in an expression product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to NHP are likely to cross-react with acorresponding mutant NHP expression product. Library clones detected viatheir reaction with such labeled antibodies can be purified andsubjected to sequence analysis according to methods well known in theart.

[0038] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721, 5,837,458, 6,117,679, and 5,723,323, which areherein incorporated by reference in their entirety.

[0039] The invention also encompasses (a) DNA vectors that contain anyof the foregoing NHP coding sequences and/or their. complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirus(hCMV) immediate early gene, regulatable, viral elements (particularlyretroviral LTR promoters), the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

[0040] Where, as in the present instance, some of the described NHPpeptides or polypeptides are thought to be cytoplasmic or nuclearproteins (although processed forms or fragments can be secreted ormembrane associated), expression systems can be engineered that producesoluble derivatives of a NHP (corresponding to a NHP extracellularand/or intracellular domains, or truncated polypeptides lacking one ormore hydrophobic domains) and/or NHP fusion protein products (especiallyNHP-Ig fusion proteins, i.e., fusions of a NHP domain to an IgFc), NHPantibodies, and anti-idiotypic antibodies (including Fab fragments) thatcan be used in therapeutic applications. Preferably, the aboveexpression systems are engineered to allow the desired peptide orpolypeptide to be recovered from the culture media.

[0041] The present invention also encompasses antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists andagonists of a NHP, as well as compounds or nucleotide constructs thatinhibit expression of a NHP sequence (transcription factor inhibitors,antisense and ribozyme molecules, or open reading frame sequence orregulatory sequence replacement constructs), or promote the expressionof a NHP (e.g., expression constructs in which NHP coding sequences areoperatively associated with expression control elements such aspromoters, promoter/enhancers, etc.).

[0042] The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant NHPs or inappropriately expressed NHPs for thediagnosis of disease. The NHP proteins or peptides, NHP fusion proteins,NHP nucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of a NHPin the body. The use of engineered host cells and/or animals can offeran advantage in that such systems allow not only for the identificationof compounds that bind to the endogenous receptor/ligand of a NHP, butcan also identify compounds that trigger NHP-mediated activities orpathways.

[0043] Finally, the NHP products can be used as therapeutics. Forexample, soluble derivatives such as NHP peptides/domains correspondingto NHPS, NHP fusion protein products (especially NHP-Ig fusion proteins,i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHPantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHP-mediated pathway) can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of soluble NHP, or a NHP-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics the,NHP could activateor effectively antagonize the endogenous NHP or a protein interactivetherewith. Nucleotide constructs encoding such NHP products can be usedto genetically engineer host cells to express such products in vivo;these genetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHP, a NHP peptide, or a NHP fusionprotein to the body. Nucleotide constructs encoding functional NHPs,mutant NHPs, as well as antisense and ribozyme molecules can also beused in “gene therapy” approaches for the modulation of NHP expression.Thus, the invention also encompasses pharmaceutical formulations andmethods for treating biological disorders.

[0044] Various aspects of the invention are described in greater detailin the subsections below.

[0045] 5.1 The NHP Sequences

[0046] The cDNA sequences and corresponding deduced amino acid sequencesof the described NHPs are presented in the Sequence Listing.

[0047] Expression analysis has provided evidence that the described NHPscan be expressed in a relatively narrow range of human tissues. Inaddition to serine-threonine kinases, the described NHPs also sharesignificant similarity to a range of additional kinase families,including kinases associated with signal transduction, from a variety ofphyla and species.

[0048] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721 and 5,837,458, which are herein incorporated byreference in their entirety.

[0049] NHP gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, worms, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate NHP transgenic animals.

[0050] Any technique known in the art may be used to introduce a NHPtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191);retrovirus mediated gene transfer into germ lines (Van der Putten etal., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting inembryonic-stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723);etc. For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115:171-229, which is incorporated byreference herein in its entirety.

[0051] The present invention provides for transgenic animals that carrythe NHP transgene in all their cells, as well as animals that carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

[0052] When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

[0053] The transgene can also be selectively introduced into aparticular cell type, thus inactivating the endogenous NHP gene in onlythat cell type, by following, for example, the teaching of Gu et al.,1994, Science, 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

[0054] Once transgenic animals have been generated, the expression ofthe recombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques that include but are not limited to Northern blot analysis oftissue samples obtained from the animal, in situ hybridization analysis,and RT-PCR. Samples of NHP gene-expressing tissue, may also be evaluatedimmunocytochemically using antibodies specific for the NHP transgeneproduct.

[0055] 5.2 NHPS and NHP Polypeptides

[0056] NHPs, NHP polypeptides, NHP peptide fragments, mutated,truncated, or deleted forms of the NHPs, and/or NHP fusion proteins canbe prepared for a variety of uses. These uses include, but are notlimited to, the generation of antibodies, as reagents in diagnosticassays, for the identification of other cellular gene products relatedto a NHP, as reagents in assays for screening for compounds that can beused as pharmaceutical reagents useful in the therapeutic treatment ofmental, biological, or medical disorders and disease. Given thesimilarity information and expression data, the described NHPs can betargeted (by drugs, oligos, antibodies, etc.) in order to treat disease,or to therapeutically augment the efficacy of therapeutic agents.

[0057] The Sequence Listing discloses the amino acid sequences encodedby the described NHP-encoding polynucleotides. The NHPs displayinitiator methionines that are present in DNA sequence contextsconsistent with eucaryotic translation initiation sites. The NHPs do notdisplay consensus signal sequences, which indicates that they may becytoplasmic or possibly nuclear proteins, although they may also besecreted or membrane associated.

[0058] The NHP amino acid sequences of the invention include the aminoacid sequences presented in the Sequence Listing as well as analoguesand derivatives thereof. Further, corresponding NHP homologues fromother species are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described above are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code is wellknown, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated .herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al., eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

[0059] The invention also encompasses proteins that are functionallyequivalent to the NHPs encoded by the presently described nucleotidesequences as judged by any of a number of criteria, including, but notlimited to, the ability to bind and modify a NHP substrate, or theability to effect an identical or complementary downstream pathway, or achange in cellular metabolism (e.g., proteolytic activity, ion flux,tyrosine phosphorylation, etc.). Such functionally equivalent NHPproteins include, but are not limited to, additions or substitutions ofamino acid residues within the amino acid sequence encoded by the NHPnucleotide sequences described above, but that result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alaninei leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0060] A variety of host-expression vector systems can be used toexpress the NHP nucleotide sequences of the invention. Where the NHPpeptide or polypeptide can exist, or has been engineered to exist, as asoluble or secreted molecule, the soluble NHP peptide or polypeptide canbe recovered from the culture media. Such expression systems alsoencompass engineered host cells that express a NHP, or functionalequivalent, in situ. Purification or enrichment of a NHP from suchexpression systems can be accomplished using appropriate detergents andlipid micelles and methods well known to those skilled in the art.However, such engineered host cells themselves may be used in situationswhere it is important not only to retain the structural and functionalcharacteristics of the NHP, but to assess biological activity, e.g., indrug screening assays.

[0061] The expression systems that may be used for purposes of theinvention include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing NHPnucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformedwith recombinant yeast expression vectors containing NHP nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing NHP sequences; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing NHP nucleotide sequences; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0062] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited, to the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouye& Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster,1989, J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target expression productcan be released from the GST moiety.

[0063] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign polynucleotidesequences. The virus grows in Spodoptera frugiperda cells. A NHP codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of NHP coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted sequence is expressed (e.g., see Smith etal., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

[0064] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the NHP nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericsequence may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a NHP product in infected hosts(e.g., see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NHP nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NHP gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NHP coding sequenceis inserted, exogenous translational control signals, including,perhaps, the ATG initiation codon, must be provided. Furthermore, theinitiation codon must be in phase with the reading frame of the desiredcoding sequence to ensure translation of the entire insert. Theseexogenous translational control signals and initiation codons can be ofa variety of origins, both natural and synthetic. The efficiency ofexpression may be enhanced by the inclusion of appropriate transcriptionenhancer elements, transcription terminators, etc. (See Bitter et al.,1987, Methods in Enzymol. 153:516-544).

[0065] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells that possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

[0066] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the NHP sequences described above can be engineered. Rather thanusing expression vectors that contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci, which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines that express the NHPproduct. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the NHP product.

[0067] A number of selection systems may be used, including but notlimited to the herpes simplex virus thymidine kinase (Wigler, et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski; 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817)genes, which can be employed in tk-, hgprt- or aprt- cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567;O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, whichconfers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc.Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol.150:1); and hygro, which confers resistance to hygromycin (Santerre, etal., 1984, Gene 30:147).

[0068] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al., allows for theready purification of non-denatured fusion proteins expressed in humancell lines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the sequence of interest is subclonedinto a vaccinia recombination plasmid such that the sequence's openreading frame is translationally fused to an amino-terminal tagconsisting of six histidine residues. Extracts from cells infected withrecombinant vaccinia virus are loaded onto Ni²⁺·nitriloaceticacid-agarose columns and histidine-tagged proteins are selectivelyeluted with imidazole-containing buffers.

[0069] Also encompassed by the present invention are fusion proteinsthat direct the NHP to a target organ and/or facilitate transport acrossthe membrane into the cytosol. Conjugation of NHPs to antibody moleculesor their Fab fragments could be used to target cells bearing aparticular epitope. Attaching the appropriate signal sequence to the NHPwould also transport the NHP to the desired location within the cell.Alternatively targeting of NHP or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in “Liposomes: A Practical Approach”, New,ed., Oxford University Press, New York and in U.S. Pat. Nos. 4,594,595,5,459,127, 5,948,767 and 6,110,490 and their respective disclosures,which are herein incorporated by reference in their entirety.Additionally embodied are novel protein constructs engineered in such away that they facilitate transport of the NHP to the target site ordesired organ, where they cross the cell membrane and/or the nucleuswhere the NHP can exert its functional activity. This goal may beachieved by coupling of the NHP to a cytokine or other ligand thatprovides targeting specificity, and/or to a protein transducing domain(see generally U.S. applications Ser. Nos. 60/111,701 and 60/056,713,both of which are herein incorporated by reference, for examples of suchtransducing sequences) to facilitate passage across cellular membranesand can optionally be engineered to include nuclear localization.

[0070] 5.3 Antibodies to NHP Products

[0071] Antibodies that specifically recognize one or more epitopes of aNHP, or epitopes of conserved variants of a NHP, or peptide fragments ofa NHP are also encompassed by the invention. Such antibodies include butare not limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0072] The antibodies of the invention can be used, for example, in thedetection of NHP in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHP. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHP expression product. Additionally, such antibodies can be usedin conjunction gene therapy to, for example, evaluate the normal and/orengineered NHP-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHP activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

[0073] For the production of antibodies, various host animals may beimmunized by injection with the NHP, a NHP peptide (e.g., onecorresponding to a functional domain of a NHP), truncated NHPpolypeptides (NHP in which oneror more domains have been deleted),functional equivalents of the NHP or mutated variant of the NHP. Suchhost animals may include but are not limited to pigs, rabbits, mice,goats, and rats, to name but a few. Various adjuvants may be used toincrease,the immunological response, depending on the host species,including but not limited to Freund's adjuvant (complete andincomplete), mineral salts such as aluminum hydroxide or aluminumphosphate, chitosan, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Alternatively, the immune response could beenhanced by combination and or coupling with molecules such as keyholelimpet hemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, choleratoxin or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0074] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497;and U.S. Pat. No. 4,376,110), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc.Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, IgD and any subclass thereof. Thehybridoma producing the mAb of this invention may be cultivated in vitroor in vivo. Production of high titers of mAbs in vivo makes this thepresently preferred method of production.

[0075] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,075,181 and5,877,397 and their respective disclosures, which are hereinincorporated by reference in their entirety. Also encompassed by thepresent invention is the use of fully humanized monoclonal antibodies asdescribed in U.S. Pat. No. 6,150,584 and respective disclosures, whichare herein incorporated by reference in their entirety.

[0076] Alternatively, techniques described for the production of. singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHP expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0077] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: the F(ab′)₂ fragments, which can be produced bypepsin digestion of the antibody molecule and the Fab fragments, whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

[0078] Antibodies to a NHP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHP, using techniques wellknown to those skilled in the art. (See, e.g., Greenspan & Bona, 1993,FASEB J 7(5):437-444; and Nissinoff, 1991, J. Immunol.147(8):2429-2438). For example antibodies that bind to a NHP domain andcompetitively inhibit the binding of NHP to its cognate receptor/ligandcan be used to generate anti-idiotypes that “mimic” the NHP and,therefore, bind, activate, or neutralize a NHP, NHP receptor, or NHPligand. Such anti-idiotypic antibodies or Fab fragments of suchanti-idiotypes can be used in therapeutic regimens involving a NHPmediated pathway.

[0079] Additionally given the high degree of relatedness of mammalianNHPs, the presently described knock-out mice (having never seen NHP, andthus never been tolerized to NHP) have a unique utility, as they can beadvantageously applied to the generation of antibodies against thedisclosed mammalian NHP (i.e., NHP will be immunogenic in NHP knock-outanimals).

[0080] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1 4 1 2301 DNA homo sapiens 1 atggccagca ccaggagtat cgagctggagcactttgagg aacgggacaa aaggccgcgg 60 ccggggtcgc ggagaggggc ccccagctcctccgggggca gcagcagctc gggccccaag 120 gggaacgggc tcatccccag tccggcgcacagtgcccact gcagcttcta ccgcacgcgg 180 accctgcagg ccctcagctc ggagaagaaggccaagaagg cgcgcttcta ccggaacggg 240 gaccgctact tcaagggcct ggtgtttgccatctccagcg accgcttccg gtcyttcgat 300 gcgctcctca tagagctcac ccgctccctgtcggacaacg tgaacctgcc ccagggtgtc 360 cgcactatct acaccatcga cggcagccggaaggtcacca gcctggacga gctgctggaa 420 ggtgagagtt acgtgtgtgc atccaatgaaccatttcgta aagtcgatta caccaaaaat 480 attaatccaa actggtctgt gaacatcaagggtgggacat cccgagcgct ggctgctgcc 540 tcctctgtga aaagtgaagt aaaagaaagtaaagatttca tcaaacccaa gttagtgact 600 gtgattcgaa gtggagtgaa gcctagaaaagccgtgcgga tccttctgaa taaaaagact 660 gctcattcct ttgaacaagt cttaacagatatcaccgaag ccattaaact agactcagga 720 gtcgtcaaga ggctctgcac cctggatggaaagcaggtta cttgtctgca agactttttt 780 ggtgatgacg atgtttttat tgcatgtggaccagaaaaat ttcgttatgc ccaagatgac 840 tttgtcctgg atcatagtga atgtcgtgtcctgaagtcat cttattctcg atcctcagct 900 gttaagtatt ctggatccaa aagccctgggccctctcgac gcagcaaatc accagcttca 960 gttaatggaa ctcccagcag ccaactttctactcctaaat ctacgaaatc ctccagttcc 1020 tctccaacta gtccaggaag tttcagaggattaaagcaga tttctgctca tggcagatct 1080 tcttccaatg taaacggtgg acctgagcttgaccgttgca taagtcctga aggtgtgaat 1140 ggaaacagat gctctgaatc atcaactcttcttgagaaat acaaaattgg aaaggtcatt 1200 ggtgatggca attttgcagt agtcaaagagtgtatagaca ggtccactgg aaaggagttt 1260 gccctaaaga ttatagacaa agccaaatgttgtggaaagg aacacctgat tgagaatgaa 1320 gtgtcaatac tgcgccgagt gaaacatcccaatatcatta tgctggtcga ggagatggaa 1380 acagcaactg agctctttct ggtgatggaattggtcaaag gtggagatct ctttgatgca 1440 attacttcgt cgaccaagta cactgagagagatggcagtg ccatggtgta caacttagcc 1500 aatgccctca ggtatctcca tggcctcagcatcgtgcaca gagacatcaa accagagaat 1560 ctcttggtgt gtgaatatcc tgatggaaccaagtctttga aactgggaga ctttgggctt 1620 gcgactgtgg tagaaggccc tttatacacagtctgtggca cacccactta tgtggctcca 1680 gaaatcattg ctgaaactgg ctatggcctgaaggtggaca tttgggcagc tggtgtgatc 1740 acatacatac ttctctgtgg attcccaccattccgaagtg agaacaatct ccaggaagat 1800 ctcttcgacc agatcttggc tgggaagctggagtttccgg ccccctactg ggataacatc 1860 acggactctg ccaaggaatt aatcagtcaaatgcttcagg taaatgttga agctcggtgt 1920 accgcgggac aaatcctgag tcacccctgggtgtcagatg atgcctccca ggagaataac 1980 atgcaagctg aggtgacagg taaactaaaacagcacttta ataatgcgct ccccaaacag 2040 aacagcacta ccaccggggt ctccgtcatcatgaacacgg ctctagataa ggaggggcag 2100 attttctgca gcaagcactg tcaagacagcggcaggcctg ggatggagcc catctctcca 2160 gttcctccct cagtggagga gatccctgtgcctggggaag cagtcccggc ccccacccct 2220 ccggaatctc ccacccccca ctgtcctcccgctgccccgg gtggtgagcg ggcaggaacc 2280 tggcgccgcc accgagactg a 2301 2 766PRT homo sapiens 2 Met Ala Ser Thr Arg Ser Ile Glu Leu Glu His Phe GluGlu Arg Asp 1 5 10 15 Lys Arg Pro Arg Pro Gly Ser Arg Arg Gly Ala ProSer Ser Ser Gly 20 25 30 Gly Ser Ser Ser Ser Gly Pro Lys Gly Asn Gly LeuIle Pro Ser Pro 35 40 45 Ala His Ser Ala His Cys Ser Phe Tyr Arg Thr ArgThr Leu Gln Ala 50 55 60 Leu Ser Ser Glu Lys Lys Ala Lys Lys Ala Arg PheTyr Arg Asn Gly 65 70 75 80 Asp Arg Tyr Phe Lys Gly Leu Val Phe Ala IleSer Ser Asp Arg Phe 85 90 95 Arg Ser Phe Asp Ala Leu Leu Ile Glu Leu ThrArg Ser Leu Ser Asp 100 105 110 Asn Val Asn Leu Pro Gln Gly Val Arg ThrIle Tyr Thr Ile Asp Gly 115 120 125 Ser Arg Lys Val Thr Ser Leu Asp GluLeu Leu Glu Gly Glu Ser Tyr 130 135 140 Val Cys Ala Ser Asn Glu Pro PheArg Lys Val Asp Tyr Thr Lys Asn 145 150 155 160 Ile Asn Pro Asn Trp SerVal Asn Ile Lys Gly Gly Thr Ser Arg Ala 165 170 175 Leu Ala Ala Ala SerSer Val Lys Ser Glu Val Lys Glu Ser Lys Asp 180 185 190 Phe Ile Lys ProLys Leu Val Thr Val Ile Arg Ser Gly Val Lys Pro 195 200 205 Arg Lys AlaVal Arg Ile Leu Leu Asn Lys Lys Thr Ala His Ser Phe 210 215 220 Glu GlnVal Leu Thr Asp Ile Thr Glu Ala Ile Lys Leu Asp Ser Gly 225 230 235 240Val Val Lys Arg Leu Cys Thr Leu Asp Gly Lys Gln Val Thr Cys Leu 245 250255 Gln Asp Phe Phe Gly Asp Asp Asp Val Phe Ile Ala Cys Gly Pro Glu 260265 270 Lys Phe Arg Tyr Ala Gln Asp Asp Phe Val Leu Asp His Ser Glu Cys275 280 285 Arg Val Leu Lys Ser Ser Tyr Ser Arg Ser Ser Ala Val Lys TyrSer 290 295 300 Gly Ser Lys Ser Pro Gly Pro Ser Arg Arg Ser Lys Ser ProAla Ser 305 310 315 320 Val Asn Gly Thr Pro Ser Ser Gln Leu Ser Thr ProLys Ser Thr Lys 325 330 335 Ser Ser Ser Ser Ser Pro Thr Ser Pro Gly SerPhe Arg Gly Leu Lys 340 345 350 Gln Ile Ser Ala His Gly Arg Ser Ser SerAsn Val Asn Gly Gly Pro 355 360 365 Glu Leu Asp Arg Cys Ile Ser Pro GluGly Val Asn Gly Asn Arg Cys 370 375 380 Ser Glu Ser Ser Thr Leu Leu GluLys Tyr Lys Ile Gly Lys Val Ile 385 390 395 400 Gly Asp Gly Asn Phe AlaVal Val Lys Glu Cys Ile Asp Arg Ser Thr 405 410 415 Gly Lys Glu Phe AlaLeu Lys Ile Ile Asp Lys Ala Lys Cys Cys Gly 420 425 430 Lys Glu His LeuIle Glu Asn Glu Val Ser Ile Leu Arg Arg Val Lys 435 440 445 His Pro AsnIle Ile Met Leu Val Glu Glu Met Glu Thr Ala Thr Glu 450 455 460 Leu PheLeu Val Met Glu Leu Val Lys Gly Gly Asp Leu Phe Asp Ala 465 470 475 480Ile Thr Ser Ser Thr Lys Tyr Thr Glu Arg Asp Gly Ser Ala Met Val 485 490495 Tyr Asn Leu Ala Asn Ala Leu Arg Tyr Leu His Gly Leu Ser Ile Val 500505 510 His Arg Asp Ile Lys Pro Glu Asn Leu Leu Val Cys Glu Tyr Pro Asp515 520 525 Gly Thr Lys Ser Leu Lys Leu Gly Asp Phe Gly Leu Ala Thr ValVal 530 535 540 Glu Gly Pro Leu Tyr Thr Val Cys Gly Thr Pro Thr Tyr ValAla Pro 545 550 555 560 Glu Ile Ile Ala Glu Thr Gly Tyr Gly Leu Lys ValAsp Ile Trp Ala 565 570 575 Ala Gly Val Ile Thr Tyr Ile Leu Leu Cys GlyPhe Pro Pro Phe Arg 580 585 590 Ser Glu Asn Asn Leu Gln Glu Asp Leu PheAsp Gln Ile Leu Ala Gly 595 600 605 Lys Leu Glu Phe Pro Ala Pro Tyr TrpAsp Asn Ile Thr Asp Ser Ala 610 615 620 Lys Glu Leu Ile Ser Gln Met LeuGln Val Asn Val Glu Ala Arg Cys 625 630 635 640 Thr Ala Gly Gln Ile LeuSer His Pro Trp Val Ser Asp Asp Ala Ser 645 650 655 Gln Glu Asn Asn MetGln Ala Glu Val Thr Gly Lys Leu Lys Gln His 660 665 670 Phe Asn Asn AlaLeu Pro Lys Gln Asn Ser Thr Thr Thr Gly Val Ser 675 680 685 Val Ile MetAsn Thr Ala Leu Asp Lys Glu Gly Gln Ile Phe Cys Ser 690 695 700 Lys HisCys Gln Asp Ser Gly Arg Pro Gly Met Glu Pro Ile Ser Pro 705 710 715 720Val Pro Pro Ser Val Glu Glu Ile Pro Val Pro Gly Glu Ala Val Pro 725 730735 Ala Pro Thr Pro Pro Glu Ser Pro Thr Pro His Cys Pro Pro Ala Ala 740745 750 Pro Gly Gly Glu Arg Ala Gly Thr Trp Arg Arg His Arg Asp 755 760765 3 2298 DNA homo sapiens 3 atggccagca ccaggagtat cgagctggagcactttgagg aacgggacaa aaggccgcgg 60 ccggggtcgc ggagaggggc ccccagctcctccgggggca gcagcagctc gggccccaag 120 gggaacgggc tcatccccag tccggcgcacagtgcccact gcagcttcta ccgcacgcgg 180 accctgcagg ccctcagctc ggagaagaaggccaagaagg cgcgcttcta ccggaacggg 240 gaccgctact tcaagggcct ggtgtttgccatctccagcg accgcttccg gtcyttcgat 300 gcgctcctca tagagctcac ccgctccctgtcggacaacg tgaacctgcc ccagggtgtc 360 cgcactatct acaccatcga cggcagccggaaggtcacca gcctggacga gctgctggaa 420 ggtgagagtt acgtgtgtgc atccaatgaaccatttcgta aagtcgatta caccaaaaat 480 attaatccaa actggtctgt gaacatcaagggtgggacat cccgagcgct ggctgctgcc 540 tcctctgtga aaagtgaagt aaaagaaagtaaagatttca tcaaacccaa gttagtgact 600 gtgattcgaa gtggagtgaa gcctagaaaagccgtgcgga tccttctgaa taaaaagact 660 gctcattcct ttgaacaagt cttaacagatatcaccgaag ccattaaact agactcagga 720 gtcgtcaaga ggctctgcac cctggatggaaagcaggtta cttgtctgca agactttttt 780 ggtgatgacg atgtttttat tgcatgtggaccagaaaaat ttcgttatgc ccaagatgac 840 tttgtcctgg atcatagtga atgtcgtgtcctgaagtcat cttattctcg atcctcagct 900 gttaagtatt ctggatccaa aagccctgggccctctcgac gcagcaaatc accagcttca 960 gttaatggaa ctcccagcag ccaactttctactcctaaat ctacgaaatc ctccagttcc 1020 tctccaacta gtccaggaag tttcagaggattaaagattt ctgctcatgg cagatcttct 1080 tccaatgtaa acggtggacc tgagcttgaccgttgcataa gtcctgaagg tgtgaatgga 1140 aacagatgct ctgaatcatc aactcttcttgagaaataca aaattggaaa ggtcattggt 1200 gatggcaatt ttgcagtagt caaagagtgtatagacaggt ccactggaaa ggagtttgcc 1260 ctaaagatta tagacaaagc caaatgttgtggaaaggaac acctgattga gaatgaagtg 1320 tcaatactgc gccgagtgaa acatcccaatatcattatgc tggtcgagga gatggaaaca 1380 gcaactgagc tctttctggt gatggaattggtcaaaggtg gagatctctt tgatgcaatt 1440 acttcgtcga ccaagtacac tgagagagatggcagtgcca tggtgtacaa cttagccaat 1500 gccctcaggt atctccatgg cctcagcatcgtgcacagag acatcaaacc agagaatctc 1560 ttggtgtgtg aatatcctga tggaaccaagtctttgaaac tgggagactt tgggcttgcg 1620 actgtggtag aaggcccttt atacacagtctgtggcacac ccacttatgt ggctccagaa 1680 atcattgctg aaactggcta tggcctgaaggtggacattt gggcagctgg tgtgatcaca 1740 tacatacttc tctgtggatt cccaccattccgaagtgaga acaatctcca ggaagatctc 1800 ttcgaccaga tcttggctgg gaagctggagtttccggccc cctactggga taacatcacg 1860 gactctgcca aggaattaat cagtcaaatgcttcaggtaa atgttgaagc tcggtgtacc 1920 gcgggacaaa tcctgagtca cccctgggtgtcagatgatg cctcccagga gaataacatg 1980 caagctgagg tgacaggtaa actaaaacagcactttaata atgcgctccc caaacagaac 2040 agcactacca ccggggtctc cgtcatcatgaacacggctc tagataagga ggggcagatt 2100 ttctgcagca agcactgtca agacagcggcaggcctggga tggagcccat ctctccagtt 2160 cctccctcag tggaggagat ccctgtgcctggggaagcag tcccggcccc cacccctccg 2220 gaatctccca ccccccactg tcctcccgctgccccgggtg gtgagcgggc aggaacctgg 2280 cgccgccacc gagactga 2298 4 765 PRThomo sapiens 4 Met Ala Ser Thr Arg Ser Ile Glu Leu Glu His Phe Glu GluArg Asp 1 5 10 15 Lys Arg Pro Arg Pro Gly Ser Arg Arg Gly Ala Pro SerSer Ser Gly 20 25 30 Gly Ser Ser Ser Ser Gly Pro Lys Gly Asn Gly Leu IlePro Ser Pro 35 40 45 Ala His Ser Ala His Cys Ser Phe Tyr Arg Thr Arg ThrLeu Gln Ala 50 55 60 Leu Ser Ser Glu Lys Lys Ala Lys Lys Ala Arg Phe TyrArg Asn Gly 65 70 75 80 Asp Arg Tyr Phe Lys Gly Leu Val Phe Ala Ile SerSer Asp Arg Phe 85 90 95 Arg Ser Phe Asp Ala Leu Leu Ile Glu Leu Thr ArgSer Leu Ser Asp 100 105 110 Asn Val Asn Leu Pro Gln Gly Val Arg Thr IleTyr Thr Ile Asp Gly 115 120 125 Ser Arg Lys Val Thr Ser Leu Asp Glu LeuLeu Glu Gly Glu Ser Tyr 130 135 140 Val Cys Ala Ser Asn Glu Pro Phe ArgLys Val Asp Tyr Thr Lys Asn 145 150 155 160 Ile Asn Pro Asn Trp Ser ValAsn Ile Lys Gly Gly Thr Ser Arg Ala 165 170 175 Leu Ala Ala Ala Ser SerVal Lys Ser Glu Val Lys Glu Ser Lys Asp 180 185 190 Phe Ile Lys Pro LysLeu Val Thr Val Ile Arg Ser Gly Val Lys Pro 195 200 205 Arg Lys Ala ValArg Ile Leu Leu Asn Lys Lys Thr Ala His Ser Phe 210 215 220 Glu Gln ValLeu Thr Asp Ile Thr Glu Ala Ile Lys Leu Asp Ser Gly 225 230 235 240 ValVal Lys Arg Leu Cys Thr Leu Asp Gly Lys Gln Val Thr Cys Leu 245 250 255Gln Asp Phe Phe Gly Asp Asp Asp Val Phe Ile Ala Cys Gly Pro Glu 260 265270 Lys Phe Arg Tyr Ala Gln Asp Asp Phe Val Leu Asp His Ser Glu Cys 275280 285 Arg Val Leu Lys Ser Ser Tyr Ser Arg Ser Ser Ala Val Lys Tyr Ser290 295 300 Gly Ser Lys Ser Pro Gly Pro Ser Arg Arg Ser Lys Ser Pro AlaSer 305 310 315 320 Val Asn Gly Thr Pro Ser Ser Gln Leu Ser Thr Pro LysSer Thr Lys 325 330 335 Ser Ser Ser Ser Ser Pro Thr Ser Pro Gly Ser PheArg Gly Leu Lys 340 345 350 Ile Ser Ala His Gly Arg Ser Ser Ser Asn ValAsn Gly Gly Pro Glu 355 360 365 Leu Asp Arg Cys Ile Ser Pro Glu Gly ValAsn Gly Asn Arg Cys Ser 370 375 380 Glu Ser Ser Thr Leu Leu Glu Lys TyrLys Ile Gly Lys Val Ile Gly 385 390 395 400 Asp Gly Asn Phe Ala Val ValLys Glu Cys Ile Asp Arg Ser Thr Gly 405 410 415 Lys Glu Phe Ala Leu LysIle Ile Asp Lys Ala Lys Cys Cys Gly Lys 420 425 430 Glu His Leu Ile GluAsn Glu Val Ser Ile Leu Arg Arg Val Lys His 435 440 445 Pro Asn Ile IleMet Leu Val Glu Glu Met Glu Thr Ala Thr Glu Leu 450 455 460 Phe Leu ValMet Glu Leu Val Lys Gly Gly Asp Leu Phe Asp Ala Ile 465 470 475 480 ThrSer Ser Thr Lys Tyr Thr Glu Arg Asp Gly Ser Ala Met Val Tyr 485 490 495Asn Leu Ala Asn Ala Leu Arg Tyr Leu His Gly Leu Ser Ile Val His 500 505510 Arg Asp Ile Lys Pro Glu Asn Leu Leu Val Cys Glu Tyr Pro Asp Gly 515520 525 Thr Lys Ser Leu Lys Leu Gly Asp Phe Gly Leu Ala Thr Val Val Glu530 535 540 Gly Pro Leu Tyr Thr Val Cys Gly Thr Pro Thr Tyr Val Ala ProGlu 545 550 555 560 Ile Ile Ala Glu Thr Gly Tyr Gly Leu Lys Val Asp IleTrp Ala Ala 565 570 575 Gly Val Ile Thr Tyr Ile Leu Leu Cys Gly Phe ProPro Phe Arg Ser 580 585 590 Glu Asn Asn Leu Gln Glu Asp Leu Phe Asp GlnIle Leu Ala Gly Lys 595 600 605 Leu Glu Phe Pro Ala Pro Tyr Trp Asp AsnIle Thr Asp Ser Ala Lys 610 615 620 Glu Leu Ile Ser Gln Met Leu Gln ValAsn Val Glu Ala Arg Cys Thr 625 630 635 640 Ala Gly Gln Ile Leu Ser HisPro Trp Val Ser Asp Asp Ala Ser Gln 645 650 655 Glu Asn Asn Met Gln AlaGlu Val Thr Gly Lys Leu Lys Gln His Phe 660 665 670 Asn Asn Ala Leu ProLys Gln Asn Ser Thr Thr Thr Gly Val Ser Val 675 680 685 Ile Met Asn ThrAla Leu Asp Lys Glu Gly Gln Ile Phe Cys Ser Lys 690 695 700 His Cys GlnAsp Ser Gly Arg Pro Gly Met Glu Pro Ile Ser Pro Val 705 710 715 720 ProPro Ser Val Glu Glu Ile Pro Val Pro Gly Glu Ala Val Pro Ala 725 730 735Pro Thr Pro Pro Glu Ser Pro Thr Pro His Cys Pro Pro Ala Ala Pro 740 745750 Gly Gly Glu Arg Ala Gly Thr Trp Arg Arg His Arg Asp 755 760 765

What is claimed is:
 1. An isolated nucleic acid molecule comprising anucleotide sequence drawn from the group consisting of SEQ ID NO:1 andSEQ ID NO:3.
 2. An isolated nucleic acid molecule comprising anucleotide sequence that: (a) encodes the amino acid sequence shown inSEQ ID NO:2; and (b) hybridizes under stringent conditions to thenucleotide sequence of SEQ ID NO:1 or the complement thereof.
 3. Anisolated nucleic acid molecule comprising a nucleotide sequence encodingthe amino acid sequence shown in SEQ ID NO:2.
 4. An isolated nucleicacid molecule comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO:4.